Planta (2003) 218: 315–322
DOI 10.1007/s00425-003-1087-3
O R I GI N A L A R T IC L E
Francisco J. L. Gordillo Æ Félix L. Figueroa
F. Xavier Niell
Photon- and carbon-use efficiency in Ulva rigida at different CO2
and N levels
Received: 18 February 2003 / Accepted: 6 July 2003 / Published online: 21 August 2003
Springer-Verlag 2003
Abstract The seaweed Ulva rigida C. Agardh (Chlorophyta) was cultured under two CO2 conditions supplied
through the air bubbling system: non-manipulated air
and 1% CO2-enriched aeration. These were also combined with N sufficiency and N limitation, using nitrate
as the only N source. High CO2 in U. rigida led to higher
growth rates without increasing the C fixed through
photosynthesis under N sufficiency. Quantum yields for
charge separation at photosystem II (PSII) reaction
centres (/PSII) and for oxygen evolution (/O2) decreased
at high CO2 even in N-sufficient thalli. Cyclic electron
flow around PSII as part of a photoprotection strategy
accompanied by decreased antennae size was suspected.
The new re-arrangement of the photosynthetic energy at
high CO2 included reduced investment in processes other
than C fixation, as well as in carbon diverted to respiration. As a result, quantum yield for new biomass-C
production (/growth) increased. The calculation of the
individual quantum yields for the different processes
involved allowed the completion of the energy flow
scheme through the cell from incident light to biomass
production for each of the CO2 and N-supply conditions
studied.
Keywords Ulva Æ Absorptance Æ Chlorophyll
fluorescence Æ Inorganic carbon Æ Nitrogen limitation Æ
Quantum yield
Abbreviations A: total thallus absorptance Æ Apig:
absorptance due to pigments Æ Astr: Absorptance due
to non-pigmented structures Æ a*: spectrally averaged in
vivo absorption cross-section of chlorophyll a Æ CCM:
carbon-concentrating mechanism Æ Chl: chlorophyll Æ
DOC: dissolved organic carbon Æ ETR: electron
F. J. L. Gordillo (&) Æ F. L. Figueroa Æ F. X. Niell
Departamento de Ecologı́a, Facultad de Ciencias,
Universidad de Málaga, Campus de Teatinos,
29071 Málaga, Spain
E-mail: [email protected]
Fax: +34-952-132000
transport rate Æ Fv/Fm: optimum quantum yield for
PSII charge separation Æ GP: gross O2 evolution rate Æ
kpig: specific light absorption coefficient for pigments Æ
kstr: specific light absorption coefficient for nonpigmented structures Æ OP: optimum O2 evolution rate Æ
PFR: photon fluence rate Æ POC: particulate organic
carbon Æ PS: photosystem Æ qN: non-photochemical
quenching Æ qP: photochemical quenching Æ /growth:
quantum yield for new biomass-C production Æ /O2:
quantum yield for gross O2 evolution Æ /PSII: quantum
yield for PSII charge separation
Introduction
One of the main queries for depicting future scenarios
for ecosystems is whether the expected increase in
atmospheric CO2 will stimulate primary production. In
aquatic environments, to answer this question it is
necessary to know not only whether photosynthesis is
saturated at the present carbon concentration in the
oceans, since many aquatic primary producers possess
carbon-concentrating mechanisms (CCMs), but also
whether there is a corresponding effect on growth
(Riebesell et al. 1993). Thus, the majority of the studies
on the effects of increasing CO2 in aquatic photosynthetic organisms have mainly focused on the uptake of
carbon through CCMs, while little attention has been
paid to other processes. This has led to a lag in knowledge
of the interaction between high CO2, light-harvesting
efficiency and nutrient assimilation in aquatic plants with
respect to their terrestrial counterparts.
For terrestrial plants, it has been proposed that the
response to elevated CO2 would be similar to that of
nitrogen limitation (Webber et al. 1994). The hypothesis
was developed and tested that elevated CO2 increases
photochemical energy use when there is a high demand
for assimilates and decreases usage when demand is low.
The acceleration of C assimilation through photosynthesis provokes a higher demand for N. If N availability
or assimilation rates cannot meet the new demand, a
316
situation equivalent to N limitation develops, and then
adaptation mechanisms often resemble those occurring
in N limitation under normal CO2 conditions. This
correspondence is not always applicable due to complex
interactions between the metabolisms of carbon and
nitrogen (e.g. Kruse et al. 2003).
Changes in carbon assimilation at elevated CO2
necessitate changes in the partitioning of absorbed
energy between heat dissipation and photochemistry in
the thylakoid membrane (Drake et al. 1997) and in the
way photochemical energy is used by the metabolism.
Previous studies on the effects of elevated CO2 on energy
partitioning in the photosynthetic apparatus have produced consistent results. Biochemically based models of
C-3 photosynthesis can be used to predict that when
photosynthesis is limited by the amount of Rubisco,
increasing atmospheric CO2 will increase light-saturated
linear electron flow through photosystem II (PSII). This
is because the stimulation of electron flow to the photosynthetic carbon reduction cycle will be greater than the
competitive suppression of electron flow to the photorespiratory carbon oxidation cycle. Then the ratio of
absorbed energy dissipated photochemically to that dissipated non-photochemically will rise (Hymus et al.
2001). In algae possessing CCMs, photorespiration is
normally lacking due to high CO2 concentration around
Rubisco even at ambient CO2 levels in the medium (Beer
et al. 2000), so that the model proposed for higher plants
is not likely to work in algae with CCMs. Information on
how CO2 will alter energy partitioning between the
processes involved in its photochemical conversion to
new biomass is very scarce for algae, and particularly so
for macroalgae. It has been previously proposed that
CO2 would somehow affect pigment synthesis (Garcı́aSánchez et al. 1994) and photosynthetic apparatus
organization (Polle et al. 2000). It seems that CO2 is
involved in the transcription of genes of the light-harvesting complex (LHC; Somanchi et al. 1998) but the
functional meaning of such regulation is unknown.
Differences in energy partitioning between photosystems
at high or low CO2 has also been reported (Satoh 2002).
Higher PSI/PSII reaction-centre ratios under low-CO2
conditions are thought to reflect the requirement of
PSI cyclic electron flow to drive the energy-expensive
carbon pump (reviewed by Kaplan and Reinhold 1999).
The flat green macroalga Ulva has often been used
as a model organism. In U. rigida growth is entirely
governed by N assimilation, while photosynthesis is
CO2-saturated at current atmospheric levels of CO2
(Björk et al. 1993; Mercado et al. 1998). In our previous work we reported that growth rate increases at
high CO2 due to photosynthesis-independent stimulation of N assimilation. The corresponding demand for
extra C is covered by: (i) reducing losses of organic C
being released to the external medium, and (ii) less
assimilated C being spent in respiration (Gordillo et al.
2001). How photon capture and energy partitioning
can meet these adaptation mechanisms has not been
tackled until now.
In this study, we attempt to quantify the quantum
yields of the main constraining processes involved in the
conversion of photons into biomass production under
different CO2 and NO3) availability situations. Quantum yield quantifies the efficiency of a light-dependent
process and can be determined for individual steps in the
photosynthetic chain of events leading to the formation
of new biomass. As a result, a complete flow-scheme of
energy partition in the cell is presented. We intend to
show that CO2 enrichment can modify not only the
photosynthetic performance but also the light-capture
strategy and the energy partitioning finally determining
the growth efficiency. Mechanisms of adaptation to
different CO2 and NO3) supply conditions are discussed.
Knowing the performance of these processes is an urgent
task since excessive growth of U. rigida in polluted
coastal areas often provokes severe environmental
problems (Sfriso 1995).
Materials and methods
Plant cultivation
Ulva rigida C. Agardh was collected in an intertidal rocky shore in
Málaga (Mediterranean Sea, Southern Spain). Healthy thalli free
from macroscopic epibiota were selected and kept for 3–4 days in
filtered (Whatman GF/F) and enriched sea water (Provasoli 1968)
at 25 C, bubbled with air at 1 l min)1, a photoperiod of 12 h:12 h
(light:darkness) and a photon fluence rate (PFR) of 100 lmol m)2
s)1 provided by white fluorescent lamps (Osram daylight L 20 W/
10 S). Light in the photosynthetically active range (PAR) range was
measured with a spherical sensor (LiCor 193 SB) connected to a
LiCor-1000 DataLogger radiometer. The indicated subsaturating
PFR was selected to avoid disagreement between measurements of
pulse amplitude modulated (PAM) fluorometry and measurements
of O2 evolution rates as observed at saturating PFRs in Ulva,
according to Beer et al. (2000), Frankling and Badger (2001), and
Longstaff et al. (2002).
Experimental design
Cultures ran for 10 days under two CO2 conditions: non-manipulated air (current atmospheric concentration: 0.035%) and CO2enriched aeration (1%); combined with two initial N concentrations
added in batch mode: N sufficiency (5 mM NO3)) and N limitation
(0.25 mM NO3), net uptake ceased after 4 days). The CO2 enrichment would correspond to 360 lM CO2, 22.51 mM HCO3), and
1.13 mM CO3) dissolved in the culture medium at equilibrium.
Thalli were cut into discs of 2 cm in diameter for cultivation.
Cultures started when 1.5 g FW were placed in an Erlenmeyer flask
containing 1 l filtered seawater (Millipore 0.22 lm) enriched with
Provasoli-based medium (Provasoli 1968). Temperature, aeration,
PFR and photoperiod were the same as for maintenance conditions described above. The water motion produced by the aeration
allowed the discs to move softly without tumbling.
The pH in CO2-enriched cultures was never below 7.7. A
control culture at pH 7.7 without CO2 enrichment eliminated any
statistically significant influence of pH on the results.
Light absorption
Chlorophyll (Chl) a, Chl b and total carotenoids were extracted
from fresh discs in N,N-dimethyl formamide for 24 h at room
temperature and total darkness. The concentrations were determined spectrophotometrically according to Wellburn (1994).
317
Total thallus absorptance for the PAR region (A) was estimated
spectrophotometrically by the opal-glass method according to
Mercado et al. (1996). The fraction of A due to pigments (Apig) and
due to structures (Astr=A)Apig) was calculated from the equations
given by Markager and Sand-Jensen (1994), except that calculations were based on thallus area instead of thallus-specific carbon
content:
A ¼ 1 eðkpig½pigþkstr w=aÞ
Apig =A ¼ kpig ½pig = kpig ½pig þ kstr w=a
Statistics
Data presented are the mean of three independent experiments,
each consisting of two cultures running in parallel for each treatment. Results were compared by two-way analyses of variance
followed by a multirange test using Fisher’s protected least
significant differences (LSD). The confidence level was set at 5%.
ð1Þ
ð2Þ
where kpig is the specific light-absorption coefficient for pigments
(m2 g)1 total pigments), [pig] is the total pigment concentration (g
m)2), kstr is the specific light-absorption coefficient for non-pigmented structures of the thallus (m2 g)1 DW) and w/a is the ratio
dry weight to area (g DW m)2).
Photosynthetic parameters
Net photosynthesis at saturating PFR (Pmax, 600 lmol m)2 s)1)
and at culture PFR (NPS, 100 lmol m)2 s)1) and dark respiration
rates were estimated by oxygen evolution using a Clark-type oxygen electrode (Yellow Spring Instruments, 5331) in a 9-ml custommade transparent Plexiglas chambers at 25 C. Rate measurements
were made at 10-min intervals. To allow the conversion from
evolved O2 to fixed CO2, the photosynthetic quotient (PQ¢) was
estimated by measuring changes in pH, simultaneously to O2 in
the photosynthesis chambers, and converted to CO2 by the
program SEAWATER (David Turner, Plymouth). Optimal quantum yield for PSII fluorescence (Fv/Fm) was measured by means
of a pulse amplitude modulated fluorimeter (PAM-2000; Waltz,
Effeltrich, Germany) after 15 min of incubation in darkness. Under
culture conditions, effective quantum yield of PSII (/PSII=DF/Fm¢ ),
as well as the photochemical (qP) and non-photochemical
quenching (qN) were also measured using the same apparatus.
According to Genty et al. (1989), Fv is the maximal variable
fluorescence of a dark-adapted sample, Fm the fluorescence intensity with all PSII reaction centres closed, F the fluorescence at
any time during induction and Fm¢ the light-saturated fluorescence.
The electron transport rate between PSII and PSI (ETR) was
calculated as:
ETR ¼ UPSII PFR 0:5 Apig
where 0.5 stands for the assumption of equal contribution of
excitons between PSI and PSII.
For clarity in the discussion of processes, we preferred to use
the PSII excitation pressure (1)qP), rather than qP. Thus, 1)qP
would indicate the proportion of reduced QA, the primary PSII
acceptor. All measurements were performed after 10 days of
cultivation.
Results
Cell contents of Chl a, Chl b and total carotenoids decreased at high CO2, especially Chl b which lost 25%
with respect to normal CO2 levels in N sufficiency
(Table 1). As a consequence, the ratio Chl b/a showed
lower values at high CO2. Nitrogen limitation also reduced the pigment content; this inhibitory effect was in
addition to that promoted by high CO2, so that thalli
grown at high CO2 and N limitation showed the poorest
pigmentation. However, this additive effect did not significantly affect the ratio Chl b/a. Changes in the spectrally averaged in vivo absorption cross-section of Chl a
(a*) denote changes in the optical properties, structure
and composition of the chloroplasts according to Berner
et al. (1989). The a* values resulted in a 50% increase at
high CO2 in N sufficiency reaching 9 m2 g)1 Chl a. This
increase was lower in N-limiting conditions.
Total pigment content was plotted against total
thallus absorptance for the PAR region (A, Fig. 1) in
order to differentiate between the absorption due to
pigments (Apig) and that due to non-pigmented structures (Astr). Data in Fig. 1 were fitted to the equation
given by Markager and Sand-Jensen (1994), described
above (Eq. 1; r=0.88, P<0.05), from which:
kpig ¼ 2:68 m2 g1 total pigments
kstr ¼ 0:014 m2 g1 DW
Table 2 shows the values of thallus total absorptance
(A) for the different treatments, as well as its two
components, Apig and Astr. In N sufficiency, A values
were slightly higher at high CO2, due to an increase in
Astr rather than Apig which remained unchanged at 0.45;
i.e., 45% of the incident light was absorbed by pigments
Total internal C and particulate organic carbon (POC)
Total internal organic C was measured with a C:H:N elemental
analyser (Perkin-Elmer 2400CHN) after drying three discs
from each culture overnight at 80 C. For POC in the culture
medium, water samples from the culture medium were filtered
(Whatman GF/F). The filter was dried overnight at 80 C and
used for determination of POC using the C:H:N elemental
analyser.
Determination of dissolved organic carbon (DOC)
Daily water samples for the determination of DOC in the medium
were analysed in an automated system (Traacs 800; Bran &
Luebbe) by means of the persulfate oxidation method which
includes UV radiation, CO2 dialysis and colorimetric determination
(Koprivnjak et al. 1995).
Table 1 Pigment composition (mg g)1 DW) and ratio, and a* values
(m2g)1 Chl a) ofUlva rigida cultured with non-enriched (Air) and
1% CO2-enriched air (+CO2), in N-sufficient (N+), and N-limited
(N)) conditions. Standard deviations in parentheses (n=6);
different superscripts indicate significant differences (P<0.05)
Parameter
Chl a
Chl b
Total
carotenoids
Chl b /a
a*
Air
+CO2
N+
N)
N+
N)
6.9 (0.7)a
5.6 (0.5)a
2.8 (0.3)a
4.3 (0.4)b
3.1 (0.3)b
1.6 (0.2)b
5.6 (0.5)c
4.2 (0.3)c
2.3 (0.2)c
2.5 (0.6)d
1.7 (0.5)d
1.0 (0.2)d
0.82 (0.03)a 0.73 (0.02)b 0.74 (0.02)b 0.70 (0.03)b
6.2 (0.6)a
5.9 (1.1)a
9.0 (1.4)b
7.6 (2.5)ab
318
Fig. 1 Relationship between total thallus absorptance (A) and
pigment concentration in Ulva rigida. Curve fitting (r=0.88,
P<0.05) corresponds to the equation given by Markager and
Sand-Jensen (see Eq. 1 in the text). Closed circles N-sufficient
samples with non-manipulated air, open circles N-limited samples
with non-manipulated air, closed squares N-sufficient samples with
1% CO2-enriched air, open squares N-limited samples with 1%
CO2-enriched air
Table 2 Quantum yields of processes involved in light absorption.
Total (A) and pigmentary (Apig) and non-pigmentary absorptances
(Astr=A)Apig), as well as optimal (Fv/Fm) and effective (/PSII) PSII
quantum yield of U. rigida cultured with non-enriched (Air) and
1% CO2-enriched air (+CO2), in N-sufficient (N+), and N-limited
(N)) conditions. Standard deviations in parentheses (n=6);
different superscripts indicate significant differences (P<0.05)
Parameter
Air
+CO2
N+
A
Apig
Astr
Fv/Fm
/PSII*
0.60
0.45
0.15
0.70
0.53
N)
(0.00)a
(0.01)a
(0.01)a
(0.00)a
(0.00)a
0.52
0.36
0.16
0.62
0.38
N+
( 0.03)b
(0.01)b
(0.01)a
(0.03)b
(0.02)b
0.64
0.45
0.19
0.73
0.40
N)
(0.02)c
(0.02)a
(0.01)b
(0.01)a
(0.01)b
0.48
0.29
0.19
0.55
0.18
(0.02)b
(0.02)c
(0.01)b
(0.05)c
(0.02)c
*Measured at PFR=100 lmol m)2 s)1
in N sufficiency, regardless the CO2 treatment. However,
the impact of N limitation on Apig was more noticeable
at high CO2 where values dropped to 0.29. The optimum
and effective quantum yield for charge separation at
PSII reaction centres (Fv/Fm and /PSII, respectively) are
also shown in Table 2. Fv/Fm remained unchanged at
high CO2 in N sufficiency, but not so /PSII, which
significantly decreased from 0.53 to 0.40 at culture PFR.
As for Apig, the influence of N limitation on decreasing
both Fv/Fm and /PSII was greater at high CO2.
Linear electron transport rates between photosystems
(ETR) are represented in Fig. 2a. The initial slope of the
curve, named the photosynthetic efficiency (a), was
affected by CO2 enrichment only under N limitation;
however, the maximum ETR (ETRmax) decreased both
due to CO2 enrichment and N limitation, so that the
lowest ETR was found at high CO2 under N limitation.
Nitrogen limitation did not promote significant differences in PSII excitation pressure (1)qP), at least at PFR
Fig. 2a–c Electron transport rates (a), PSII excitation pressure (b),
and non-photochemical quenching (c) at increasing PFR of U.
rigida cultured at different CO2 and N-supply conditions.
Closed circles N-sufficient samples with non-manipulated air,
open circles N-limited samples with non-manipulated air, closed
squares N-sufficient samples with 1% CO2-enriched air, open
squares N-limited samples with 1% CO2-enriched air
below 300 lmol m)2 s)1 (Fig. 2b). Increased CO2 led to
higher excitation pressure regardless of the N conditions
to near saturation beyond 300 lmol m)2 s)1. Both N
limitation and increased CO2 led to an early saturation
of energy dissipation measured as qN (Fig. 2c) with
respect to the control conditions as PFR increased. Final
qN values at saturating irradiance were similar for all
culture conditions except for high CO2 + N limitation.
The fate of the carbon fixed through photosynthesis
is represented as a percentage relative to the control
culture (air, N+) in Table 3. The level of CO2 conditioned both the total value and the relative proportion of
the different processes involved. At low CO2 the impact
of N limitation was only 5%, while at high CO2 the
reduction respect to N sufficiency was almost 20%. The
percentage of C diverted to respiration was markedly
reduced at high CO2 under both N conditions. At high
319
Table 3 Comparative carbon use by U. rigida under different CO2
and N supply situations expressed as a percentage of the total
carbon fixed by the control culture (air, N+). Samples were
cultured for 10 days with non-enriched (Air) and 1% CO2-enriched
air (+CO2), in N-sufficient (N+), and N-limited (N)) conditions.
Standard deviations in parentheses (n=6); different superscripts
indicate significant differences (P<0.05)
Air
+CO2
N+
Respiration
POC
DOC
Growth
Total
N)
a
73.84 (2.67)
4.54 (0.15)a
2.94 (0.1)a
18.68 (2.34)a
100
N+
a
75.62 (2.66)
5.07 (0.16)b
4.09 (0.13)b
10.68 (3.56)b
95.46
Table 4 Quantum yields for oxygen evolution based on light
absorbed by pigment (/O2=mol O2:mol photons); and quantum
yields for net growth (/growth=mol C:mol photons) based on
incident light (i), total absorbed light (A) and light absorbed by
pigments (pig) in U. rigida. Samples were cultured for 10 days with
non-enriched (Air) and 1% CO2-enriched air (+CO2), in N-sufficient (N+), and N-limited (N)) conditions. Standard deviations in
parentheses (n=6); different superscripts indicate significant
differences (P<0.05)
N)
b
34.7 (1.78)
4.8 (0.29)a
2.76 (0.17)a
39.15 (3.01)c
81.4
Air
c
40.92 (0.89)
4.63 (0.12)a
5.6 (0.13)c
11.56 (4.45)b
62.72
Fig. 3 Energy distribution of incident light among different
processes as it flows from the thallus surface (top) to the production
of new biomass-C (bottom), expressed as a percentage. a Transmitted through; b absorbed by non-pigmented structures; c
dissipated in the photosynthetic antennae; d suboptimal O2
production, assuming the optimal to be eight electrons per gross
O2 evolved; e photosynthetic energy available for processes others
than C fixation; f fixed carbon lost by respiration; g, h organic
carbon released and found in the medium in particulate and
dissolved form, respectively; i fixed carbon invested in new biomass
production
CO2 in N sufficiency, the percentage invested in growth
was the highest, despite total C fixed being lower than
the control. N limitation led to the lowest investment of
C into new biomass regardless of the CO2 conditions.
The energy flow through the cell can be quantified
and represented as in Fig. 3. The total height of the
column represents the total amount of photons by units
of area exposed (1/2 of total thallus area) that reached
the thallus during the culture period. This energy
suffered different losses before getting to form new
biomass carbon. The first loss of energy, neglecting
N+
/O2
Ugrowth
i
Ugrowth
A
Ugrowth
pig
+CO2
N)
0.118
0.106
0.021 (0.006)a 0.012 (0.004)a
0.034 (0.009)a 0.023 (0.008)a
0.045 (0.012)a 0.034 (0.012)a
N+
N)
0.086
0.197
0.044 (0.009)b 0.013 (0.005)a
0.069 (0.014)b 0.027 (0.010)a
0.098 (0.022)b 0.044 (0.017)a
reflectance, is that not being absorbed by the thallus and
then transmitted trough [a, (1)A)·100]. This was higher
in N limitation. Part of the energy was absorbed by nonpigmented structures [b, (A)Apig)·100], the rest being
absorbed by pigments. Light absorbed by pigments
decreased by N limitation but was not affected by CO2.
Of that energy, part was dissipated as a radiationless
decay from the antennae in their way to the reaction
centres {c, [(1)/PSII) Apig]·100}. This loss was slightly
higher at high CO2. The box d represents suboptimal
conversion of excitons to oxygen evolved, assuming an
optimal conversion of eight electrons per O2 (i.e.
/O2=0.125 O2 per photon), according to the Z-scheme
for photosynthesis {[1)(GP/OP) /PSII Apig]·100}, where
GP is gross photosynthesis (i.e. measured gross O2
evolution rate) and OP the optimum photosynthesis (i.e.
maximum O2 that should have been produced if operating at eight electrons per O2). Then, this represents the
disagreement between the observed O2 evolution in the
photosynthetic chambers and that calculated according
to the number of photons estimated to reach the reaction centres divided by eight. Under normal (nonenriched) CO2 conditions the electron demand per
oxygen evolved was close to the optimum, with values of
8.5 and 9.3 for N-sufficient and N-limited thalli,
respectively (i.e. /O2 of 0.118 and 0.106, respectively;
Table 4). CO2 enrichment altered this step in different
ways depending on N conditions. Under N sufficiency
more than 11 electrons were needed per O2 evolved,
whereas under N limitation a controversial value of 5
was estimated. The box e in Fig. 3 represents the
amount of photosynthetic energy remaining after carbon
fixation. This remaining photosynthetic energy would be
available for other processes such as CCMs, nutrient
uptake and biosynthesis of macromolecules. In Fig. 3,
box e is calculated then as {[(GP/OP) /PSII Apig]·100 )
(f+g+h+i)}. This portion of energy decreased both
with CO2 enrichment and with N limitation. Box f
represents the portion of photosynthetic energy invested
in fixing C that was lost in the respiration process [(Dark
resp./OP) /PSII Apig · 100]. Respiration markedly
320
decreased at high CO2, especially under N sufficiency.
Box g is the portion corresponding to fixed carbon released to the external medium and found in particulate
form [(POC/OP) /PSII Apig · 100]; box h is the percentage corresponding to fixed carbon released to the
external medium and found in dissolved form [(DOC/
OP) /PSII Apig · 100]. Finally, box i represents the percentage of incident energy that was invested in fixing
carbon to form new biomass, i.e. net growth [(total
internal carbon in the new biomass/OP) /PSII Apig ·
100). This portion decreased under N-limiting conditions for both CO2 situations but it increased at high
CO2 when N was supplied in excess.
Table 4 shows the quantum
yields for net growth
growth
based
on
incident
light
U
; total absorbed
light
i
growth
growth
and light absorbed by pigments Upig
:
UA
Results indicate that provided N is not limiting, the
efficiency in the use of light is practically double at high
CO2 with respect to non-manipulated-air conditions.
When quantum yield is expressed in terms of light absorbed by pigments, growth efficiency got to 0.098, close
to the theoretical maximum of 0.125.
Discussion
The portion of incident light absorbed by non-pigmented
structures (Astr) was quite significant for all treatments.
This enforces the suggestion that this factor should be
taken into account in order to determine the proportion
of incident light that is actually absorbed by the antennae
pigments (Apig). Uncertainties on such estimations would
lead to a limited knowledge of ETR as measured by pulse
amplitude modulated fluorometry, as previously discussed by Beer et al. 2000 and Frankling and Badger
2001; however, very few studies on ETR include this sort
of measurement. This is due, at least in part, to the difficulty of performing good estimates in species with thick
thallus structure. Another assumption remains: we have
ignored the portion of light being back-scattered,
although this is likely to be less than 1% under a perpendicular light beam (Frost-Christensen and SandJensen 1992). The values of A measured agreed with
those reported by Frankling and Badger (2001) for U.
australis, and Astr was close to the value given by
Markager and San-Jensen (1994) of 0.13 for U. lactuca.
Changes in pigment composition triggered by increased CO2 are a frequently observed response. Garcı́aSánchez et al. (1994) and Nakano et al. (1997) reported a
significant decrease in Chl a concentration in the red
macroalga Gracilaria tenuistipitata and in rice leaves,
respectively, in a CO2-enriched environment; other
plants, such as the seagrass Zostera marina (Zimmerman
et al. 1997), the green microalga Scenedesmus obtusiusculus (Larsson et al. 1985) and the brown alga Fucus
serratus (Johnston and Raven 1990) did not show any
change in their pigment concentration. A hypothesis has
been previously suggested according to which the
decrease in pigment content at high CO2 would correspond to the elimination of molecules that are in excess
and would not participate efficiently in the light capture
for photosynthesis, after a new rearrangement of the
light-harvesting system as part of the acclimation strategy. This has been called ‘‘the pigment economy
hypothesis’’ (Gordillo et al. 1999). This is supported by
the fact that the optimal quantum yield for charge separation at PSII reaction centres (Fv/Fm) and the initial
slope of the ETR–PFR curves are the same at high and
low CO2 (in N sufficiency). A complementary hypothesis
suggests that lower pigment content at high CO2 could
be an adaptation reducing the antenna size to avoid
over-excitation of electron transport, although this
might not affect the electron transport chain itself (Saxe
et al. 1998). This is in agreement with the decrease in the
Chlb/a ratio and the increase in a* observed. Similar
results have been found for the unicellular chlorophyte
Dunaliella viridis (Gordillo et al. 2003) and the cyanobacterium Spirulina platensis (Gordillo et al. 1999) under
equivalent culture conditions.
Differences in ETRmax can be attributed to differences in the amount of Rubisco present in the chloroplast. The fall in final values of the ETR curves when
either increasing CO2 or limiting N coincided with the
fall in the pool of soluble proteins reported previously
(Gordillo et al. 2001), of which Rubisco constitutes a
majority. The increase in excitation pressure (1)qP)
observed at high CO2 (Fig. 2b) indicates the establishment of a mechanism of over-excitation of the quinone
pool that was not provoked by an increase in the
amount of charge separations at PSII, since ETR was
even lower at high CO2. Cyclic flow around PSII as a
photoprotective resource is a possible explanation, as
discussed below. The increase in non-photochemical
quenching is one of the first responses to environmental
stress. In U. rigida, both high CO2 and N limitation
increased qN at intermediate PFRs (Fig. 2c), evidencing
an early establishment of energy dissipation processes.
Values of /O2 close to the optimum of 0.125 measured under control CO2 conditions were in agreement
with those found by Beer et al. (2000) for U. lactuca and
U. fasciata. Frankling and Badger (2001) found that
closeness to optimal values was CO2-dependent in
U. australis and other macroalgae. We found that up to
11 electrons were needed to produce 1 O2 molecule at
high CO2 in N-sufficient thalli. According to Frankling
and Badger (2001), ETR in excess of O2 evolution in
Ulva is unlikely to represent higher photorespiration
rates or Mehler activity. The alternative explanation
given by these authors is cyclic electron flow around
PSII from the quinone acceptor QB (Pheophytin) via
cyt b559. In the case where electrons cannot be resupplied to P680+ from the water oxidation complex, the
strong oxidant P680+ can be re-reduced by cyt b559,
preventing the oxidation of nearby elements of the
reaction centre. This mechanism would provide
additional photoprotective capacity for keeping PSII
active without O2 evolution or photosynthetic energy
321
production. Lavaud et al. (2002) found that up to three
extra excitons would be needed at PSII for O2 evolution,
preventing both acceptor-side photoinhibition in oxygen-evolving PSII and donor-side photoinhibition when
the oxygen-evolving complex is temporarily inactivated.
The 11 electrons needed per O2 and higher reduction
status of the QA (1)qP,) observed at high CO2 (Fig. 2b)
would support this interpretation. Another reason for a
deviation from the optimum at high CO2 (in N sufficiency) could be the difference in light-capture efficiency
by PSI and PSII. CO2 has been reported to affect lightcapture efficiency of PSI to a larger extent than that
of PSII (Satoh et al. 2002). Bürger et al. (1988) and
Miyachi et al. (1996) found in some cyanobacteria and
unicellular green algae that both the ratio PSI/PSII
and /O2 decreased in cells grown at high CO2. The
higher PSI/PSII under low CO2 conditions is thought to
reflect the demand of the excess energy from PSI for
cyclic electron flow to drive the CCMs, as previously
suggested by Ogawa and Ogren (1985). When both high
CO2 and N limitation conditions were simultaneously
applied, a controversial value of /O2=0.2 was derived.
It is known that nitrogen limitation also has differential
effects on photosynthetic efficiency of PSI and PSII
(Berges et al. 1996). Therefore, since all calculations of
/O2 assume equal distribution of photons between PSI
and PSII according to the Z-scheme (four photons
each), further investigations are needed to elucidate
whether this assumption can be made without significant
error.
The main difference between higher plants and algae
possessing CCMs with respect to elevated CO2 is the
way in which the use of photochemical energy is altered.
In higher plants, elevated CO2 partial pressure leads to
decreased photorespiration rates, and then concomitant
increased carboxylation efficiency is observed (e.g.
Habash et al. 1995). However, many algae, among them
Ulva, have virtually no photorespiration at current CO2
levels (Beer et al. 2000) due to the existence of energetically expensive CCMs which increase the level of CO2
around Rubisco. When exposed to CO2-enriched conditions, the expression and activity of CCMs are generally repressed (Björk et al. 1993; Gordillo et al. 2001)
and the energy normally being invested in such concentrating processes is made available to other processes
(Raven 1991). That could be the case for N-sufficient
U. rigida. Both the portion of photosynthetic energy
available for processes other than carboxylation (box e
in Fig. 3) and the energy from respiration (box f in
Fig. 3) decreased markedly. That being the case,
photosynthetic CO2 fixation does not necessarily increase at high CO2, whilst cell growth (box i in Fig. 3)
reaches higher efficiency (i.e. higher /growth). A question
remains whether CCMs at control CO2 conditions
would use energy from photosynthesis or from respiration. Huertas et al. (2002) have recently demonstrated
that the source of energy for active CO2 transport across
the membrane comes from photosynthetic electron
transport in the unicellular chlorophyte Nannochlorys
atomus, a species that, unlike U. rigida (Björk et al.
1992), lacks an active HCO3) transporter and extracellular carbonic anhydrase.
At high CO2, the photosynthetic energy not invested
in C fixation or CCMs could well serve for nitrate
assimilation. U. rigida is a nitrophilic organism and
growth stimulation at high CO2 is known to be governed
by an increase in N assimilation (Gordillo et al. 2001),
i.e. a higher portion of energy has to be invested in nitrate transport and reduction. In the C4 sorghum,
Watling et al. (2000) found that the ratio /CO2 to /PSII
was lower in plants grown at elevated CO2, implying
increased electron transport to acceptors other than
CO2.
In conclusion, elevated CO2 changed the energy capture and partitioning characteristics of U. rigida, such
that acclimation to high CO2 implied a different pattern
of energy flow through the cell mainly characterised by
decreased energy demands for CCMs and lower respiration rates, leading to a more efficient use of photons
for growth, despite lower /O2. The latter was possibly a
consequence of photoprotection of PSII reaction centres
by suspected cyclic flow around PSII accompanied by
decreased antenna size.
Acknowledgements This work was financed by the Spanish Ministry of Science and Technology Project REN 2002-00340/MAR, and
the Andalusian research group RNM0176.
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